Integrated 13C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli

The use of parallel labeling experiments for ¹³C metabolic flux analysis (¹³C-MFA) has emerged in recent years as the new gold standard in fluxomics. The methodology has been termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. In this contribution, we have tested the limits of COMPLETE-MFA by demonstrating integrated analysis of 14 parallel labeling experiments with Escherichia coli. An effort on such a massive scale has never been attempted before. In addition to several widely used isotopic tracers such as [1,2-¹³C]glucose and mixtures of [1-¹³C]glucose and [U-¹³C]glucose, four novel tracers were applied in this study: [2,3-¹³C]glucose, [4,5,6-¹³C]glucose, [2,3,4,5,6-¹³C]glucose and a mixture of [1-¹³C]glucose and [4,5,6-¹³C]glucose. This allowed us for the first time to compare the performance of a large number of isotopic tracers. Overall, there was no single best tracer for the entire E. coli metabolic network model. Tracers that produced well-resolved fluxes in the upper part of metabolism (glycolysis and pentose phosphate pathways) showed poor performance for fluxes in the lower part of metabolism (TCA cycle and anaplerotic reactions), and vice versa. The best tracer for upper metabolism was 80% [1-¹³C]glucose+20% [U-¹³C]glucose, while [4,5,6-¹³C]glucose and [5-¹³C]glucose both produced optimal flux resolution in the lower part of metabolism. COMPLETE-MFA improved both flux precision and flux observability, i.e. more independent fluxes were resolved with smaller confidence intervals, especially exchange fluxes. Overall, this study demonstrates that COMPLETE-MFA is a powerful approach for improving flux measurements and that this methodology should be considered in future studies that require very high flux resolution.     

Optimal tracers for parallel labeling experiments and 13C metabolic flux analysis: A new precision and synergy scoring system

¹³C-Metabolic flux analysis (¹³C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by ¹³C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for ¹³C-MFA. The best single tracers were doubly ¹³C-labeled glucose tracers, including [1,6-¹³C]glucose, [5,6-¹³C]glucose and [1,2-¹³C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6-¹³C]glucose and [1,2-¹³C]glucose. Combined analysis of [1,6-¹³C]glucose and [1,2-¹³C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1-¹³C]glucose +20% [U-¹³C]glucose.     

COMPLETE-MFA: Complementary parallel labeling experiments technique for metabolic flux analysis

We have developed a novel approach for measuring highly accurate and precise metabolic fluxes in living cells, termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. The COMPLETE-MFA method is based on combined analysis of multiple isotopic labeling experiments, where the synergy of using complementary tracers greatly improves the precision of estimated fluxes. In this work, we demonstrate the COMPLETE-MFA approach using all singly labeled glucose tracers, [1-¹³C], [2-¹³C], [3-¹³C], [4-¹³C], [5-¹³C], and [6-¹³C]glucose to determine precise metabolic fluxes for wild-type Escherichia coli. Cells were grown in six parallel cultures on defined medium with glucose as the only carbon source. Mass isotopomers of biomass amino acids were measured by gas chromatography–mass spectrometry (GC–MS). The data from all six experiments were then fitted simultaneously to a single flux model to determine accurate intracellular fluxes. We obtained a statistically acceptable fit with more than 300 redundant measurements. The estimated flux map is the most precise flux result obtained thus far for E. coli cells. To our knowledge, this is the first time that six isotopic labeling experiments have been successfully integrated for high-resolution ¹³C-flux analysis.     

Evidence for transketolase-like TKTL1 flux in CHO cells based on parallel labeling experiments and 13C-metabolic flux analysis

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. It provides precursors for the biosynthesis of nucleotides and contributes to the production of reducing power in the form of NADPH. It has been hypothesized that mammalian cells may contain a hidden reaction in PPP catalyzed by transketolase-like protein 1 (TKTL1) that is closely related to the classical transketolase enzyme; however, until now there has been no direct experimental evidence for this reaction. In this work, we have applied state-of-the-art techniques in ¹³C metabolic flux analysis (¹³C-MFA) based on parallel labeling experiments and integrated flux fitting to estimate the TKTL1 flux in CHO cells. We identified a set of three parallel labeling experiments with [1-¹³C]glucose+[4,5,6-¹³C]glucose, [2-¹³C]glucose+[4,5,6-¹³C]glucose, and [3-¹³C]glucose+[4,5,6-¹³C]glucose and developed a new method to measure ¹³C-labeling of fructose 6-phosphate by GC-MS that allows intuitive interpretation of mass isotopomer distributions to determine key fluxes in the model, including glycolysis, oxidative PPP, non-oxidative PPP, and the TKTL1 flux. Using these tracers we detected a significant TKTL1 flux in CHO cells at the stationary phase. The flux results suggest that the main function of oxidative PPP in CHO cells at the stationary phase is to fuel the TKTL1 reaction. Overall, this study demonstrates for the first time that carbon atoms can be lost in the PPP, by means other than the oxidative PPP, and that this loss of carbon atoms is consistent with the hypothesized TKTL1 reaction in mammalian cells.     

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the ¹³C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-¹³C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the ¹³C enrichment. The approach to correct for these effects in metabolic flux studies using δ¹³C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and ¹³C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of ¹³C in proteinogenic amino acids over the whole range of applied concentrations of [1-¹³C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in ¹³C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.     

Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by 13C metabolic flux analysis

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no ¹³C-flux studies on xylose metabolism. In the absence of experimentally validated flux maps, constraint-based approaches such as MOMA and RELATCH cannot be used to guide new metabolic engineering designs. In this work, we have addressed this critical gap in current understanding by performing comprehensive characterizations of glucose and xylose metabolism under aerobic and anaerobic conditions, using recent state-of-the-art techniques in ¹³C metabolic flux analysis (¹³C-MFA). Specifically, we quantified precise metabolic fluxes for each condition by performing parallel labeling experiments and analyzing the data through integrated ¹³C-MFA using the optimal tracers [1,2-¹³C]glucose, [1,6-¹³C]glucose, [1,2-¹³C]xylose and [5-¹³C]xylose. We also quantified changes in biomass composition and confirmed turnover of macromolecules by applying [U-¹³C]glucose and [U-¹³C]xylose tracers. We demonstrated that under anaerobic growth conditions there is significant turnover of lipids and that a significant portion of CO₂ originates from biomass turnover. Using knockout strains, we also demonstrated that β-oxidation is critical for anaerobic growth on xylose. Quantitative analysis of co-factor balances (NADH/FADH₂, NADPH, and ATP) for different growth conditions provided new insights regarding the interplay of energy and redox metabolism and the impact on E. coli cell physiology.     

Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for 13C metabolic flux analysis

In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and ¹³C-metabolic flux analysis (¹³C-MFA). Here, cells were grown in parallel cultures with [1-¹³C]glucose and [U-¹³C]glucose as tracers and ¹³C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of ¹³C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for ¹³C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased ¹³C-flux measurements in C. acetobutylicum.     

The biosynthesis of the antioxidant caffeic acid derivative subulatin by the liverwort Jungermannia subulata cultured in vitro

The in vitro cultured liverwort Jungermannia subulata produces the unique molecule subulatin. In this study, we examined the incorporation of [1-¹³C] and [1,2-¹³C₂] glucose, [2-¹³C] arabinose, [2-¹³C] caffeic acid, and [1-¹³C] phenylalanine into subulatin. The trilobatinoic acid C unit of subulatin incorporated ¹³C atoms from [1-¹³C] and [1,2-¹³C₂] glucose and from [2-¹³C] arabinose but not from any other of the other precursors. Based on these results and labeling patterns, the trilobatinoic acid C unit of subulatin appears to be biosynthesized from arabinose-5-phosphate and phosphoenolpyruvate.     

Carbohydrate and Amino Acid Metabolism in the Ectomycorrhizal Ascomycete Sphaerosporella brunnea during Glucose Utilization : A C NMR Study

Nuclear magnetic resonance spectroscopy was utilized to study the metabolism of [1-¹³C]glucose in mycelia of the ectomycorrhizal ascomycete Sphaerosporella brunnea. The main purpose was to assess the biochemical pathways for the assimilation of glucose and to identify the compounds accumulated during glucose assimilation. The majority of the ¹³C label was incorporated into mannitol, while glycogen, trehalose and free amino acids were labeled to a much lesser extent. The high enrichment of the C1/C6 position of mannitol indicated that the polyol was formed via a direct route from absorbed glucose. Randomization of the ¹³C label was observed to occur in glucose and trehalose leading to the accumulation of [1,6-¹³C]trehalose and [1,6-¹³C]glucose. This suggests that the majority of the glucose carbon used to form trehalose was cycled through the metabolically active mannitol pool. The proportion of label entering the free amino acids represented 38% of the soluble ¹³C after 6 hours of continuous glucose labeling. Therefore, amino acid biosynthesis is an important sink of assimilated carbon. Carbon-13 was incorporated into [3-¹³C]alanine and [2-¹³C]-, [3-¹³C]-, and [4-¹³C]glutamate and glutamine. From the analysis of the intramolecular ¹³C enrichment of these amino acids, it is concluded that [3-¹³C]pyruvate, arising from [1-¹³C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase, and pyruvate carboxylase (or phosphoenolpyruvate carboxykinase). Intramolecular ¹³C labeling patterns of glutamate and glutamine were similar and are consistent with the operation of the Krebs cycle. There is strong evidence for (a) randomization of the label on C2 and C3 positions of oxaloacetate via malate dehydrogenase and fumarase, and (b) the dual biosynthetic and respiratory role of the citrate synthase, aconitase, and isocitrate dehydrogenase reactions. The high flux of carbon through the carboxylation (presumably pyruvate carboxylase) step indicates that CO₂ fixation is an important component of the carbon metabolism in S. brunnea, and it is likely that this anaplerotic role is particularly prevalent during NH₄⁺ assimilation. The most relevant information resulting from this investigation is (a) the occurrence of the mannitol cycle, (b) a large part of the trehalose pool is synthesized after the cycling of glucose-carbon through the mannitol cycle, and (c) pyruvate (or phosphoenolpyruvate) carboxylation plays an important role in the primary metabolism of glucose-fed mycelia.     

Effects of Potassium and Glutamine on Metabolism of Glucose in Astrocytes

The metabolic effects of extracellular glutamine (2.5 mM) or high potassium (25 mM) on glucose metabolism were studied in cultured cerebellar astrocytes. High potassium caused an increased glycolytic flux and an increase in glutamine release. Exposure to glutamine increased glycolytic flux and alanine formation, indicating that glutamine uptake is an energy requiring process. The effects of glutamine and high potassium on glycolytic flux were additive. Formation of metabolites from [1-¹³C]glucose and [2-¹³C]acetate confirmed the effects of glutamine and high potassium on glycolytic metabolism. In the presence of extracellular glutamine, analysis of the ¹³C labeling patterns of citrate and glutamine indicated a decrease in the cycling ratio and/or pyruvate carboxylation and glutamine synthesis from [1-¹³C]glucose did occur, but was decreased. Exposure to high potassium led to extracellular accumulation of acetate, presumably through non-enzymatic decarboxylation of pyruvate.     

13C metabolic flux analysis of the extremely thermophilic,fast growing,xylose-utilizing Geobacillus strain LC300

Thermophiles are increasingly used as versatile hosts in the biotechnology industry. One of the key advantages of thermophiles is the potential to achieve high rates of feedstock conversion at elevated temperatures. The recently isolated Geobacillus strain LC300 grows extremely fast on xylose, with a doubling time of less than 30 min. In the accompanying paper, the genome of Geobacillus LC300 was sequenced and annotated. In this work, we have experimentally validated the metabolic network model using parallel ¹³C-labeling experiments and applied ¹³C-metabolic flux analysis to quantify precise metabolic fluxes. Specifically, the complete set of singly labeled xylose tracers, [1-¹³C], [2-¹³C], [3-¹³C], [4-¹³C], and [5-¹³C]xylose, was used for the first time. Isotopic labeling of biomass amino acids was measured by gas chromatography mass spectrometry (GC–MS). Isotopic labeling of carbon dioxide in the off-gas was also measured by an on-line mass spectrometer. The ¹³C-labeling data was then rigorously integrated for flux elucidation using the COMPLETE-MFA approach. The results provided important new insights into the metabolism of Geobacillus LC300, its efficient xylose utilization pathways, and the balance between carbon, redox and energy fluxes. The pentose phosphate pathway, glycolysis and TCA cycle were found to be highly active in Geobacillus LC300. The oxidative pentose phosphate pathway was also active and contributed significantly to NADPH production. No transhydrogenase activity was detected. Results from this work provide a solid foundation for future studies of this strain and its metabolic engineering and biotechnological applications.     

Metabolic flux ratio analysis by parallel 13C labeling of isoprenoid biosynthesis in Rhodobacter sphaeroides

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from ¹³C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-¹³C]- and [4-¹³C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer ¹³C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.     

Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.     

Cortical metabolism in pyruvate dehydrogenase deficiency revealed by ex vivo multiplet C NMR of the adult mouse brain

The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the ¹³C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-¹³C₂]glucose (oxidized by both neurons and glia) and [1,2-¹³C₂]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by ¹³C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of ¹³C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The ¹³C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-¹³C]acetate relative to [1,6-¹³C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron–glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.     

Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-¹³C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-¹³C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the ¹³C NMR spectra due to loss of the label as ¹³CO₂. Uptake of [3-¹³C]pyruvate by the cells was considerably slower than with [2-¹³C]acetate; however, the labelling patterns were similar with the exception of increased [3-¹³C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-¹³C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine.     

Re-examination of metabolic fluxes in <Emphasis Type="Italic">Escherichia coli</Emphasis> during anaerobic fermentation of glucose using <Superscript>13</Superscript>C labeling experiments and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy

Improved design of metabolic flux estimation using mixed label ¹³C labeling experiments and identifiability analysis motivated re-examination of metabolic fluxes during anaerobic fermentation in the Escherichia coli. Comprehensive metabolic flux maps were determined by using a mixture of differently labeled glucose and compared to conventional flux maps obtained using extracellular measurements and comprehensive metabolic flux maps obtained using only U-¹³C glucose as the substrate. As expected, conventional flux analysis performs poorly in comparison to ¹³C-MFA, especially in the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Identifiability analysis indicated and experiments confirmed that a mixture of 10% U-^l3C glucose, 25% 1-¹³C glucose, and 65% naturally labeled glucose significantly improved the statistical quality of all calculated fluxes in the PP pathway, the EMP pathway, the anaplerotic reactions, and the tricarboxylic acid cycle. Modifying the network topology for the presence and absence of the Entner-Doudoroff pathway and the glyoxylate shunt did not affect the value or quality of estimated fluxes significantly. Extracellular measurement of formate production was necessary for the accurate estimation of the fluxes around the formate node.     

Metabolic Effects of Blocking Lactate Transport in Brain Cortical Tissue Slices Using an Inhibitor Specific to MCT1 and MCT2

A novel inhibitor of lactate transport, AR-C122982, was used to study the effect of inhibiting the monocarboxylate transporters MCT1 and MCT2 on cortical brain slice metabolism. We studied metabolism of l-[3-¹³C]lactate, and d-[1-¹³C]glucose under a range of conditions. Experiments using l-[3-¹³C]lactate showed that the inhibitor AR-C122982 altered exchange of lactate. Under depolarizing conditions, net flux of label from d-[1-¹³C]glucose was barely altered by 10 or 100 nM AR-C122982. In the presence of AMPA or glutamate there were increases in net flux of label and metabolic pool sizes. These data suggest lactate may supply compartments in the brain not usually directly accessed by glucose. In general, it would appear that movement of lactate between cell types is not essential for metabolic activity, with the heavy metabolic workloads imposed being unaffected by inhibition of MCT1 and MCT2. Further experiments investigating the mechanism of operation of AR-C122982 are necessary to corroborate this finding.     

13C and 31P NMR studies on sugar metabolism in Bombyx mori and Philosamia cynthia ricini larvae

Studies were made on ¹³C and ³¹P NMR in larvae of two species of silkworm, Bombyx mori and Philosamia cynthia ricini, in vivo as well as in vitro to determine the pathways of glucose utilization, especially those to amino acids as components of silk fibroin. Results showed that the ¹³C of [1-¹³C]glucose administered orally into 5th instar larvae of both species was incorporated into glucose-1-phosphate, glucose-6-phosphate and trehalose. Serine, glutamate, glutamine, citrate, malate, trehalose and sorbitol-6-phosphate were detected in the hemolymphs of these larvae as metabolites of [1-¹³C]glucose. Two days after [1-¹³C]glucose administration, labeled alanine, glycine, serine, urea, glycogen, trehalose and glycerol were clearly detected in Bombyx larvae. Starvation caused rapid consumption of administered [1-¹³C]glucose with very little accumulation of ¹³C in glycogen or trehalose. In the in vivo³¹P NMR spectra of Bombyx larvae, ATP, arginine phosphate, sorbitol-6-phosphate, uridine diphosphoglucose, phosphoenolpyruvate and inorganic phosphate were detected with some sugar phosphates, such as glucose-1-phosphate and glucose-6-phosphate. During starvation, the intensity of the signal of inorganic phosphate increased and those of sugar phosphate other than sorbitol-6-phosphate decreased, but these changes were reversed by oral administration of glucose.     

Metabolism of the fast-growing bacterium Vibrio natriegens elucidated by 13C metabolic flux analysis

Vibrio natriegens is a fast-growing, non-pathogenic bacterium that is being considered as the next-generation workhorse for the biotechnology industry. However, little is known about the metabolism of this organism which is limiting our ability to apply rational metabolic engineering strategies. To address this critical gap in current knowledge, here we have performed a comprehensive analysis of V. natriegens metabolism. We constructed a detailed model of V. natriegens core metabolism, measured the biomass composition, and performed high-resolution ¹³C metabolic flux analysis (¹³C-MFA) to estimate intracellular fluxes using parallel labeling experiments with the optimal tracers [1,2−¹³C]glucose and [1,6−¹³C]glucose. During exponential growth in glucose minimal medium, V. natriegens had a growth rate of 1.70 1/h (doubling time of 24 min) and a glucose uptake rate of 3.90 g/g/h, which is more than two 2-fold faster than E. coli, although slower than the fast-growing thermophile Geobacillus LC300. ¹³C-MFA revealed that the core metabolism of V. natriegens is similar to that of E. coli, with the main difference being a 33% lower normalized flux through the oxidative pentose phosphate pathway. Quantitative analysis of co-factor balances provided additional insights into the energy and redox metabolism of V. natriegens. Taken together, the results presented in this study provide valuable new information about the physiology of V. natriegens and establish a solid foundation for future metabolic engineering efforts with this promising microorganism.